The long term goal of this project is to determine the role of phosphorylation in the regulation of chicken progesterone receptor (PR) function. PR is a member of a superfamily of ligand activated transcription factors including the other steroid receptors, Vitamin D, retinoic acid, and thyroid receptors as well as other proteins whose ligand if any has not yet been identified. Recent studies have shown that phosphorylation is a key mechanism for regulating the activity of transcription factors. We have identified 5 phosphorylation sites in the receptor which can be divided into three classes--l. Sites which are partially phosphorylated, but whose phosphorylation increases in response to progesterone administration. 2. Sites phosphorylated only in response to progesterone administration. 3. Sites which are constitutively phosphorylated. The specific aims for the next grant period are first, to determine the role of individual phosphorylations in PR function by analysis of receptor site mutants. Glycine, which will mimic the dephosphorylated state, and aspartic acid which may mimic the phosphorylated state, will be substituted for serine. The effect of these mutations on transcription, DNA binding and interactions with other proteins will be examined to determine the role of each phosphorylation site. Second, additional constitutive phosphorylation sites will be identified using the methods used for the hormone dependent sites and the role of the sites studied as described above. Finally, most analyses of steroid hormone receptor function have been performed using receptor from tissue which has not been treated with hormone. Numerous studies have shown that hormone administration results in increased steroid receptor phosphorylation and it is presumably this form which is active in vivo. Therefore, we will purify receptor from progesterone treated animals, and the activities of this modified receptor will be compared to receptor from untreated animals. These studies will provide a good understanding of the role of phosphorylation in PR function. Since PR belongs to a superfamily of transcription factors, the information obtained will provide a basis for understanding and testing the role of phosphorylation of the related regulatory proteins.